*Target (protein/gene name): Thymidylate synthase *NCBI Gene # or RefSeq#:3282062/NC_002946.2
*Protein ID (NP or XP #) or Wolbachia#: YP_208417.1 (XP or NP number not found) *Organism (including strain): Neisseria gonorrhoeae (strain ATCC 700825/FA 1090) Etiologic Risk Group (see link below):http://sti.bmj.com/content/early/2014/10/13/sextrans-2014-051670.short */Disease Information (sort of like the Intro to your Mini Research Write up):Neisseria gonorrhoeae is a Gram-negative bacterial species that causes the sexually transmitted disease gonorrhea. The bacteria is spread from person to person sexual contact and uses pili to attach to proteins in the sperm. Symptoms of gonorrhea include burning when urinating, yellow, green, or white discharge, and painful bowel movements. Gonorrhea is very common in the United States, as it has about 3 million cases per year; however, the disease can be treated by antibiotics such as Azithromycin that are prescribed by a medical professional. Link to TDR Targets page (if present): n/a Link to Gene Database page (NCBI):https://www.ncbi.nlm.nih.gov/gene/?term=3282062
Essentiality of this protein:
The synthesis of thymidylic acid is essential, because it is the only source of thymidine and it is crucial for DNA replication in every organism.
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer Complex of proteins?: 1 amino acid chain and 1 ligand in ASU Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): https://www.ebi.ac.uk/chembl/target/inspect/CHEMBL5328
*EC#: 2.1.1.45 Link to BRENDA EC# page:http://www.brenda-enzymes.org/enzyme.php?ecno=2.1.1.45 -- Show screenshot of BRENDA enzyme mechanism schematic Fig 1. BRENDA enzyme mechanism schematic for the protein thymidylate synthase. The enzyme reacts with a compound dUMP and creates compounds dihydrofolate and dTMP. Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Protein assays were used to determine protein concentrations in PBMC cytosolic lysates to locate thymidylate synthase activity. -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information https://link.springer.com/article/10.1007/s00216-012-6676-1 -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
-MTHF salt from Merck Eprova (Schaffhausen, Switzerland) $76
-Ficollpaque (TM) PLUS from General Electric Healthcare Little Chalfont, UK) $197.24 - $675.30
-Phosphate-buffered saline (PBS) from GIBCO BRL (Gaithersberg, MD, USA) $19.97, 500 mL, pH of 7.4
-Tris-HCl from Roche (Woerden, the Netherlands) $142.00
-EDTA, MgCl2•6H20, DL-dithiotreitol (DTT), NaF, 2-metraptoethanol (BME), carbon, 2'-deoxyuridine 5' monophosphate (dUMP) from Sigma (St. Louis, MO, USA)
-5-3H-dUMP with specific activity of 0.555-1.11 TBq/mmol from Moravek (Brea, CA, USA)
-Dextra T500 from Pharmacia (Uppsula, Sweden) $116.00, 5g
-Ultima Gold from Canberra Packard (Meridan, CT, USA) $154.00 (1x1 L)
Structure (PDB or Homology model) Fig. 2 Image of Homologous Protein (4LRR) complex between E.coli Thymidylate Synthase, dUMP, and F9. Protein is taken from organism Escherichia coli.
-- PDB # or closest PDB entry if using homology model: 4LRR
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB Fig. 3 Pairwise alignment of 4LRR_A from BLASTP search in NCBI.
---- Query Coverage: n/a
---- Max % Identities: 100%
---- % Positives: 100%
---- Chain used for homology: A
Current Inhibitors: Fluorouracil Expression Information (has it been expressed in bacterial cells):
The drug has been put in E. coli. Purification Method: n/a Image of protein (PyMol with features delineated and shown separately): Fig. 4 Image of 1RXC protein E. Coli uridine phosphorylase, with 5-fluorouracil ribose-1-phosphate complex *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSKSDVFHLGLTKNDLQGATLAIVPGDPDRVEKIAALMDKPVKLASHREFTTWRAELDGKPVIVCSTGIG
GPSTSIAVEELAQLGIRTFLRIGTTGAIQPHINVGDVLVTTASVRLDGASLHFAPLEFPAVADFECTTAL
VEAAKSIGATTHVGVTASSDTFYPGQERYDTYSGRVVRHFKGSMEEWQAMGVMNYEMESATLLTMCASQG
LRAGMVAGVIVNRTQQEIPNAETMKQTESHAVKIVVEAARRLL
*length of your protein in Amino Acids: 253 aa Molecular Weight of your protein in kiloDaltons using the**Expasy ProtParam**website: 27159.08 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 17085 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. Fig. 5 TMpred graph image with amino acid sequence of 1RXC protein in E. coli organism. *CDS Gene Sequence (paste as text only):
ATGTCCAAGTCTGATGTTTTTCATCTCGGCCTCACTAAAAACGATTTACAAGGGGCTACGCTTGCCATCG
TCCCTGGCGACCCGGATCGTGTGGAAAAGATCGCCGCGCTGATGGATAAGCCGGTTAAGCTGGCATCTCA
CCGCGAATTCACTACCTGGCGTGCAGAGCTGGATGGTAAACCTGTTATCGTCTGCTCTACCGGTATCGGC
GGCCCGTCTACCTCTATTGCTGTTGAAGAGCTGGCACAGCTGGGCATTCGCACCTTCCTGCGTATCGGTA
CAACGGGCGCTATTCAGCCGCATATTAATGTGGGTGATGTCCTGGTTACCACGGCGTCTGTCCGTCTGGA
TGGCGCGAGCCTGCACTTCGCACCGCTGGAATTCCCGGCTGTCGCTGATTTCGAATGTACGACTGCGCTG
GTTGAAGCTGCGAAATCCATTGGCGCGACAACTCACGTTGGCGTGACAGCTTCTTCTGATACCTTCTACC
CAGGTCAGGAACGTTACGATACTTACTCTGGTCGCGTAGTTCGTCACTTTAAAGGTTCTATGGAAGAGTG
GCAGGCGATGGGCGTAATGAACTATGAAATGGAATCTGCAACCCTGCTGACCATGTGTGCAAGTCAGGGC
CTGCGTGCCGGTATGGTAGCGGGTGTTATCGTTAACCGCACCCAGCAAGAGATCCCGAATGCTGAGACGA
TGAAACAAACCGAAAGCCATGCGGTGAAAATCGTGGTGGAAGCGGCGCGTCGTCTGCTGTAA *GC% Content for gene: 54.068241% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): n/a *GC% Content for gene (codon optimized): n/a
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
*NCBI Gene # or RefSeq#: 3282062/NC_002946.2
*Protein ID (NP or XP #) or Wolbachia#: YP_208417.1 (XP or NP number not found)
*Organism (including strain): Neisseria gonorrhoeae (strain ATCC 700825/FA 1090)
Etiologic Risk Group (see link below): http://sti.bmj.com/content/early/2014/10/13/sextrans-2014-051670.short
*/Disease Information (sort of like the Intro to your Mini Research Write up): Neisseria gonorrhoeae is a Gram-negative bacterial species that causes the sexually transmitted disease gonorrhea. The bacteria is spread from person to person sexual contact and uses pili to attach to proteins in the sperm. Symptoms of gonorrhea include burning when urinating, yellow, green, or white discharge, and painful bowel movements. Gonorrhea is very common in the United States, as it has about 3 million cases per year; however, the disease can be treated by antibiotics such as Azithromycin that are prescribed by a medical professional.
Link to TDR Targets page (if present): n/a
Link to Gene Database page (NCBI): https://www.ncbi.nlm.nih.gov/gene/?term=3282062
Essentiality of this protein:
The synthesis of thymidylic acid is essential, because it is the only source of thymidine and it is crucial for DNA replication in every organism.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer
Complex of proteins?: 1 amino acid chain and 1 ligand in ASU
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
https://www.ebi.ac.uk/chembl/target/inspect/CHEMBL5328
*EC#: 2.1.1.45
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.1.1.45
-- Show screenshot of BRENDA enzyme mechanism schematic
Fig 1. BRENDA enzyme mechanism schematic for the protein thymidylate synthase. The enzyme reacts with a compound dUMP and creates compounds dihydrofolate and dTMP.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Protein assays were used to determine protein concentrations in PBMC cytosolic lysates to locate thymidylate synthase activity.
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
https://link.springer.com/article/10.1007/s00216-012-6676-1
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
-MTHF salt from Merck Eprova (Schaffhausen, Switzerland) $76
-Ficollpaque (TM) PLUS from General Electric Healthcare Little Chalfont, UK) $197.24 - $675.30
-Phosphate-buffered saline (PBS) from GIBCO BRL (Gaithersberg, MD, USA) $19.97, 500 mL, pH of 7.4
-Tris-HCl from Roche (Woerden, the Netherlands) $142.00
-EDTA, MgCl2•6H20, DL-dithiotreitol (DTT), NaF, 2-metraptoethanol (BME), carbon, 2'-deoxyuridine 5' monophosphate (dUMP) from Sigma (St. Louis, MO, USA)
-5-3H-dUMP with specific activity of 0.555-1.11 TBq/mmol from Moravek (Brea, CA, USA)
-Dextra T500 from Pharmacia (Uppsula, Sweden) $116.00, 5g
-Ultima Gold from Canberra Packard (Meridan, CT, USA) $154.00 (1x1 L)
Structure (PDB or Homology model)
Fig. 2 Image of Homologous Protein (4LRR) complex between E.coli Thymidylate Synthase, dUMP, and F9. Protein is taken from organism Escherichia coli.
-- PDB # or closest PDB entry if using homology model: 4LRR
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Fig. 3 Pairwise alignment of 4LRR_A from BLASTP search in NCBI.
---- Query Coverage: n/a
---- Max % Identities: 100%
---- % Positives: 100%
---- Chain used for homology: A
Current Inhibitors: Fluorouracil
Expression Information (has it been expressed in bacterial cells):
The drug has been put in E. coli.
Purification Method: n/a
Image of protein (PyMol with features delineated and shown separately):
Fig. 4 Image of 1RXC protein E. Coli uridine phosphorylase, with 5-fluorouracil ribose-1-phosphate complex
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSKSDVFHLGLTKNDLQGATLAIVPGDPDRVEKIAALMDKPVKLASHREFTTWRAELDGKPVIVCSTGIG
GPSTSIAVEELAQLGIRTFLRIGTTGAIQPHINVGDVLVTTASVRLDGASLHFAPLEFPAVADFECTTAL
VEAAKSIGATTHVGVTASSDTFYPGQERYDTYSGRVVRHFKGSMEEWQAMGVMNYEMESATLLTMCASQG
LRAGMVAGVIVNRTQQEIPNAETMKQTESHAVKIVVEAARRLL
*length of your protein in Amino Acids: 253 aa
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 27159.08 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 17085
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Fig. 5 TMpred graph image with amino acid sequence of 1RXC protein in E. coli organism.
*CDS Gene Sequence (paste as text only):
ATGTCCAAGTCTGATGTTTTTCATCTCGGCCTCACTAAAAACGATTTACAAGGGGCTACGCTTGCCATCG
TCCCTGGCGACCCGGATCGTGTGGAAAAGATCGCCGCGCTGATGGATAAGCCGGTTAAGCTGGCATCTCA
CCGCGAATTCACTACCTGGCGTGCAGAGCTGGATGGTAAACCTGTTATCGTCTGCTCTACCGGTATCGGC
GGCCCGTCTACCTCTATTGCTGTTGAAGAGCTGGCACAGCTGGGCATTCGCACCTTCCTGCGTATCGGTA
CAACGGGCGCTATTCAGCCGCATATTAATGTGGGTGATGTCCTGGTTACCACGGCGTCTGTCCGTCTGGA
TGGCGCGAGCCTGCACTTCGCACCGCTGGAATTCCCGGCTGTCGCTGATTTCGAATGTACGACTGCGCTG
GTTGAAGCTGCGAAATCCATTGGCGCGACAACTCACGTTGGCGTGACAGCTTCTTCTGATACCTTCTACC
CAGGTCAGGAACGTTACGATACTTACTCTGGTCGCGTAGTTCGTCACTTTAAAGGTTCTATGGAAGAGTG
GCAGGCGATGGGCGTAATGAACTATGAAATGGAATCTGCAACCCTGCTGACCATGTGTGCAAGTCAGGGC
CTGCGTGCCGGTATGGTAGCGGGTGTTATCGTTAACCGCACCCAGCAAGAGATCCCGAATGCTGAGACGA
TGAAACAAACCGAAAGCCATGCGGTGAAAATCGTGGTGGAAGCGGCGCGTCGTCTGCTGTAA
*GC% Content for gene: 54.068241%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): n/a
*GC% Content for gene (codon optimized): n/a
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**